The research objective of this project is to investigate the interactions of adeno-associated virus (AAV) with its host cell. The underlying hypothesis is that by understanding these interactions as they apply to the biology of the virus we can contribute to the use of AAV vectors for gene therapy. Staff members focus on two types of interactions: those involved in viral transduction of the target cell, and those between the Rep proteins of the wild type AAV and their cellular partners. Current projects study the tropism and transduction pathways of AAV serotypes, the lifecycle of these viral isolates, and identify and functionally characterize novel cellular proteins that interact with the AAV Rep proteins. One project the Unit has focused on is the characterization of two new AAV serotypes, AAV4 and AAV5. These new viral isolates are being studied both as natural mutations of other serotypes for understanding the biology of this genus of virus and because of their unique cell tropism, as novel vectors for gene transfer. In a large part as a result of our work AAV4 and AAV5 are now accepted as useful vectors for gene transfer and are actively being evaluated in several gene therapy applications including gene transfer to the lung, CNS, eye, and salivary gland. We believe that just as our characterization of AAV4 and AAV5 have advanced the field of gene therapy, the development of new vectors also will have an impact on other gene therapy applications as well as our understanding of parvovirus biology. As a first step in this endeavor, we have developed a PCR based method for rapidly identifying and cloning divergent AAV isolates (Katano 2004). We have recently utilitzed this system to clone a new AAV serotype from a bovine adenovirus sample termed bovine AAV (BAAV) (Schmidt 2004). Our characterization of this virus and one isolated by direct cloning from an avian adenovirus (Bossis 2003) represents a beginning of this project. However, we have already identified aspects unique to each of their replication mechanisms and specifically demonstrated that BAAV can efficiently transduce hair cells in the inner ear, an important target cell for the treatment of deafness and balance disorders (Di Pasquale submitted). Our knowledge of the interactions necessary for cellular transduction with AAV4 and AAV5 clearly has been helpful in defining their biology and likely will identify novel applications for these vectors. Furthermore, the tools we have developed while examining AAV4 and AAV5 tropism can be applied to other novel vectors we have isolated. We expect this information will aid in identifying optimal target cells for the different AAV vectors and also aid in the development of a new generation of vectors for gene transfer with very defined cell tropism. In the future we plan to further refine our tools for studying interactions necessary for cellular transduction, identify critical interactions in the transduction pathway, identify the domains critical for these interaction on the virus surface, as well as to identify new AAV isolates that maybe useful for gene therapy applications.